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My lab studies evolution of bacteria in novel environments, and in particular the evolutionary potential lurking in the proteome due to inefficient side activities of enzymes that normally serve other functions. Current work focuses on determining how such “promiscuous” activities have been patched together into a pathway for degradation of pentachlorophenol, a toxic anthropogenic pollutant, and how the bacterium manages to survive the toxicity caused by pentachlorophenol and its degradation products. Other projects address how bacteria can adapt to deletion of an essential gene and the process by which new enzymes arise from promiscuous enzymes by a process of gene duplication and divergence.

Current Research

Enzymes are superb catalysts, capable of accelerating reactions by up to 26 orders of magnitude. The conditions required to produce and to use enzymes are “green” – they do not require toxic organic solvents and/or high temperatures and pressures that are costly in terms of energy usage.

Most microbes contain an impressive 1000-2000 enzymes. However, a change in the environment may require new enzymes. A frequent source of new enzymes is a pre-existing enzyme with a promiscuous activity; promiscuous activities are accidental side activities that arise from the highly reactive environments at enzyme active sites. When a promiscuous enzyme is recruited to do a new job, it is often inefficient. Further, there may be an “adaptive conflict” between the original and newly important activities; mutations that improve the new function often damage the old function. This conundrum is typically resolved by a process of gene duplication followed by divergence of one copy to encode an efficient new enzyme while the other copy continues to encode the original enzyme (see Figure 1).

A current project in the lab addresses evolution of a new enzyme using a model system in which a gene encoding an essential enzyme (ArgC) has been deleted. Another enzyme (ProA) present in the bacterium has a promiscuous secondary activity that corresponds to that of ArgC, but it is too inefficient to rescue growth. However, a single mutation allows the enzyme to serve both functions, albeit poorly. This “weak-link” enzyme limits growth of the bacterium; thus, the cells are under strong selective pressure for emergence of clones in which the levels of one or both enzyme activities are improved.

Using this model system in the bacterium Escherichia coli, we have found that some clones accumulate 50 copies of a genomic region containing the gene encoding the weak-link enzyme. We are currently developing a method that will allow us to follow the appearance of mutations in individual gene copies and thereby to follow the detailed dynamics of the process depicted in Fig. 1.

When we investigated this model system in a different bacterium, Salmonella enterica, we found – to our surprise - that gene amplification did not occur. Rather, we found clones with a number of point mutations, most of which simply increased the concentration of the weak-link enzyme in the cells. This finding suggests that S. enterica lacks the capacity to undergo the process shown in Fig. 1.

The insights emerging from this work suggest that only some microbes in a community will be able to meet the challenge of a changing environment by evolving new enzymes. We are beginning to understand the roles of important factors such as the repertoire of enzymes encoded by the genome, the nature of available promiscuous activities, and the location of genes encoding promiscuous activities in the genome. These findings have important implications for understanding how microbial communities respond to novel environmental conditions such as the presence of pesticides and industrial pollutants.

The Innovation-Amplification-Divergence model for evolution of a new enzyme (neo-B) starting from a gene encoding an enzyme with a primary activity A and a weak secondary activity b that becomes useful after a change in environment. Image: Shelley Copley